Optimized lymphocyte subsets by CD7 and CD33

Two problems have remained at identification and quantification of lymphocyte subsets: the incomplete removal of non-lymphoid cells from the lymphocyte gate and the lack of a restricted marker to identify NK cells. By replacing well-established CD14 by CD33 and CD16/CD56 by CD7 we expected to remove even basophils and CD14- monocyte subsets from the gate and to avoid misclassification of CD16+ respectively CD56+ monocytes.
Conclusion:
The advantage of CD33 over CD14 at the creation of a pure lymphocyte gate could be demonstrated. CD33 enables the exclusion of all monocyte subpopulations as well as basophils and granulocytes. CD7 and CD16/56 exhibited as equivalent in various CD33 settings, whereas the mean proportion of single CD56+ NK cells was significantly lower. As CD7 is restricted to T cells and NK cells in peripheral blood, misclassification of contaminating monocytes is avoided and CD7+ NK cells can be identified by lack of CD3. Use of CD33/CD7 causes a mean purity of about 99.5% within the revised lymphocyte gate. High inclusivity and high purity can be achieved concurrently, so the comparability of reference values may be increased. We propose the adoption of CD7 and CD33 for the quantification of lymphocyte subsets. Compensation was accomplished automatically by use of Flow-Set™ Fluorospheres (Beckman Coulter, Inc., Brea, CA USA) and single-stained tubes containing CD45 in FITC respectively in PE, ECD, PE-CY5 or PE-CY7 and subsequent verification by a combination of CD4/CD8/CD45/CD3/CD19 (Beckman Coulter, Inc., Brea, CA USA). Fine tuning was done manually. Threshold was set on FSC (100). Flow cytometer performance was checked using Flow-Check™ Fluorospheres (Beckman Coulter, Inc., Brea, CA USA).